Abstract The objective of this proposal is to develop a series of specific biothiol probes that will exhibit different ratiometric spectroscopic properties after undergoing reversible reactions, and thus quantitatively monitor the dynamics of biothiols through real-time imaging with subcellular resolution. Despite the existence of myriad small molecule fluorescent probes developed for biological imaging, very few can provide meaningful quantitative results, especially when tasked to detect redox signaling molecules, like glutathione (GSH) and H2S. Our recent work demonstrated that reversibility of sensing reactions is key to quantitatively monitoring the dynamics of small molecules in cells. Ratiometric probes are preferred for live cell imaging because they allow quantitative measurements of analyte concentrations independent of probe concentration. Taking advantage of reversible Michael additions, we developed CouBro, the first fluorescent probe for quantitative imaging of GSH in live cells. Due to the reversible nature of the reaction between the probe and GSH, we are able to quantify mM concentrations of GSH with as little as 50 nM CouBro. Furthermore, the GSH concentrations in several cell lines, measured using CouBro, are well correlated with those values obtained from lysates. In addition, we showed that this live imaging method has excellent reproducibility and is able to detect GSH fluctuations in cells upon external stimulation. In the preliminary study, we developed a computational chemistry approach to predict the thermodynamics and kinetics of reactions between biothiols and their probes, which will guide our design of biothiol probes. We also developed organelle specific H2S probes by applying genetically encoded protein technology to reaction-based small molecule fluorescent probes. This universal targeting strategy enables us to infer the signaling molecule concentration in the micro-environment around a protein of interest. In Aim 1, we will develop a series of GSH probes with fast kinetics and organelle specificity to monitor intracellular GSH dynamics. The probe design process will be facilitated by computational chemistry. In Aim 2, we will develop new reversible chemistry for H2S specific reactions. Due to inconsistently reported H2S levels, ranging from nM to M, H2S probes with a range of dissociation constants will be developed. We will also monitor H2S signaling dynamics by labeling key enzymes responsible for H2S production and proteins specific to certain organelles. In Aim 3, we will apply these newly developed biothiol probes to investigate Grx3 mediated GSH metabolism and its interplay with H2S signaling in cancer cells, particularly during tumorigenesis in vivo. Successful completion of this project will provide a comprehensive toolbox for quantitative imaging of GSH and H2S dynamics and further elucidate their roles in redox-related cancer signaling and development.